The circadian clock regulates a wide array of behavioral, physiological and biochemical phenomena in eukaryotes. In fact, dysfunction of the circadian clock can lead to severe medical disorders in man. In Drosophila, mutational analyses have identified several genes that are important for clock function. The broad objective of this project is to determine how two of these genes, period (per) and restless (rls), contribute to the molecular mechanisms that underlie circadian clock function. The per gene appears to function within the circadian pacemaker. An important aspect of this function may be related to the ability of per protein to regulate the circadian synthesis of its own mRNA (i.e the per feedback loop). One aim of this project is to determine how circadian cycling of per mRNA levels is regulated and whether this cycling is important for circadian behavior. Initially, in vivo and in vitro mapping experiments will be used to identify sequences and factors that regulate per mRNA cycling. These studies will establish whether per protein is acting to regulate transcription at the DNA level or through another factor. The role per mRNA cycling plays in regulating circadian behavior will be assessed by determining how behavior is influenced by inappropriately timed per protein expression and noncyclic expression of per mRNA . The relationship between per mRNA cycling and the circadian pacemaker will be examined by determining whether per mRNA cycling (which is itself a free running circadian rhythm) can function autonomously or is always correlated with a functioning circadian pacemaker in the brain. The rls gene acts downstream of per in the circadian clock. Another aim of this project is to determine how rls functions to output information from the pacemaker and whether per plays a role in regulating rls function. The transposable element that disrupts rls gene activity will be used as an entry point for isolating this gene. DNA fragments which rescue rls arrhythmicity (and therefore contain the rls gene) will be used to isolate rls cDNA's. Nucleic acid and antibody probes will be used to determine the temporal and spatial expression of rls, including any differences in expression due to the presence of upstream (per) mutations. The rls protein sequence will be analyzed for the presence of conserved sequence motifs indicative of its localization, structure or function. Mosaic mapping studies will be used to map the rls expressing cells relevant to locomotor activity output. The molecular mechanisms defined by these studies may be of general importance since locomotor activity rhythms are under clock control in many organisms.